Comparison of PLZF Gene Expression between Pluripotent Stem Cells and Testicular Germ Cells

Objective Spermatogonial stem cells (SSCs), as unipotent stem cells, are responsible for the production of sperm throughout the male’s life. Zinc finger and BTB domain containing 16 (ZBTB16/PLZF) genes provide various functions in the cell development, signaling pathway, growth regulatory and differentiation. Here, we aimed to investigate expression of the PLZF germ cell gene marker in testis, SSCs, pluripotent embryonic stem cells (ES cells) and ES-like cells of mouse testis. Materials and Methods In this experimental study, we examined the expression of the PLZF germ cell marker in the testis section and testicular cell culture of neonate and adult mice by immunohistochemistry (IMH), immunocytochemistry (ICC) and Fluidigm Real-Time polymerase chain reaction (PCR). Results IMH data indicated that the PLZF protein was localized in the neonate testis cells of the tubules center as well as the basal compartment of adult testis seminiferous tubules. Counting PLZF IMH-positive cells in the sections of seminiferous tubules of adult and neonate testis revealed significant expression of positive cells in adult testis compared to the neonate (P<0.05). Under in vitro conditions, isolated SSC colonies were strongly ICC-positive for the PLZF germ cell marker, while ES cells and ES-like cells were negative for PLZF. Fluidigm Real-Time-PCR analysis demonstrated a significant expression of the PLZF germ cell gene in the neonate and adult SSCs, compared to ES cells and ES-like cells (P<0.05). Conclusion These results indicate that PLZF is a specific transcription factor of testicular germ cell proliferation, but it is down- regulated in pluripotent germ cells. This can be supportive for the analysis of germ cells development both in vitro and in vivo.


Introduction
Germ cells are formed and matured during early embryogenesis from primordial germ cells (PGCs) (1). Spermatogonial stem cells (SSCs) are the adult stem cells located in the basal membrane of seminiferous tubules of testis. They receive cytokines from somatic cells including Sertoli cells, blood vessels, Leydig cells and macrophages. SSCs can be isolated by fluorescence-activated cell sorting (FACS), magnetic-activated cell sorting (MACS), matrix selection and morphology-based selection (2)(3)(4). SSCs have the potential for conversion into embryonic stem (ES)-like pluripotent stem cells under defined in vitro culture conditions (2)(3)(4)(5).
Extrinsic secreted growth factors from the SSCs niche and intrinsic gene expression play a crucial role in the maintenance of SSCs (2,6). Extrinsic factors which are produced and secreted by Sertoli cells include glial cellderived neurotrophic factor (GDNF) and KIT ligand (KITL) (7). Intrinsic factors include PLZF (8,9), ETV5 (10), Taf4b (11), Bcl6b (12), Pou5f1, Nrg1, Nanog and Gja1 (13)(14)(15) as well as Gfra1 and RET (16). The transcription factor PLZF, as a transcriptional repressor that regulates the epigenetic state of undifferentiated cells, is involved in different cellular functions such as cell proliferation, apoptosis and differentiation during spermatogenesis, neurogenesis and embryonic development (8,17,18). Filipponi et al. (19) demonstrated that PLZF directly represses the transcription of kit, a marker of spermatogonial differentiation. PLZF plays an essential role in the selfrenewal and maintenance of the SSC in the testis niche (8). It has been shown that PLZF is co-expressed with Oct4 in undifferentiated spermatogonia. It has also been demonstrated that loss of the encoding PLZF gene produces limited numbers of normal spermatozoa and then leading progressively to the lack of respected germline after birth. During embryogenesis, PLZF regulates the stage of gene expressions of limb and axial skeletal patterning (8,9,20). During limb development, it has been demonstrated that PLZF has genetic relationship with Gli3 and Hox5 genes (21,22). Previous studies showed that PLZF was expressed in testis and SSCs, therefore recognized as a SSC marker (23)(24)(25). In the present research we have extended our study to the expression of PLZF marker in the neonate and adult testis sections, isolated SSCs, ES cells and generated ES-like cells from mouse testicular culture to evaluate if PLZF has the same expression pattern in both testicular germ cells and pluripotent stem cells. The results indicated that PLZF is clearly expressed in germ cells, but not in pluripotent stem cells.

Culture of the embryonic stem and ES-like cells
ES and ES-like cell lines were originated from our previous study (2). These cells were cultured in medium with KO-DMEM, composed of 1% NEAA solution, 15% FBS, 1% L-glutamine, 0.1% β-mercaptoethanol, LIF at a final concentration of 1000 U/ml and 1% Pen/Strep (2).

Gene expression analyses on the Fluidigm Biomark system
Quantity of the PLZF gene expression (Mm01176868_ m1) in the neonate SSCs, adult SSCs, ES cells, and ES-like cells were examined by dynamic array chips (Fluidigm). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH, Mm99999915_g1) was used as housekeeping gene for normalization. Cultured cells were selected with a micromanipulator, lysed with lysis buffer solution containing 1.3 μl TE buffer, 0.2 μl RT/Taq Superscript III (Invitrogen, USA), 9 μl RT-PreAmp Master Mix, 5.0 μl Cells Direct 2× Reaction Mix (Invitrogen, USA), and 2.5 μl 0.2× assay pool. Using TaqMan real-time PCR on the BioMark Real-Time quantitative PCR (qPCR) system, the amount of RNAtargeted copies was evaluated. Samples were examined in two technical repeats. The Ct values were analyzed by GenEx software from the MultiD analysis (2, 3, 6).

Immunocytochemical staining
SSCs, ES cells and ES-like cells were fixed with 4% paraformaldehyde and then permeabilized with 0.1% Triton/PBS. Cells were blocked with 1% BSA/PBS and followed by incubation with primary antibody PLZF. In the next step, we used overnight incubation fluorochrome species-specific secondary antibody and the labeled cells were nuclear counterstained with 0.2 µg/ml of 4', 6-diamidino-2-phenylindole (DAPI) dye. The labeled positive cells were studied with a confocal microscope Zeiss LSM 700 (Germany), and images were acquired using a Zeiss LSM-TPMT camera (Germany) (2,(26)(27)(28).

Tissue processing for immunohistofluorescence staining
Mouse testis tissue was washed with PBS and fixed in 4% paraformaldehyde. Dehydrated tissue was surrounded in Paraplast Plus and cut with a microtome machine at 10 µm thickness. Testis tissue sections were mounted on Superfrost Plus slides and kept at room temperature until used. For processing of immunohistofluorescence staining, samples were washed with xylene followed by gradually replacing with water in ethanol before staining. For the tissue sections, antigen retrieval was performed by heat-induced epitope retrieval at 95˚C for 20 minutes, non-specific binding site of tissue samples was blocked with 10% serum/0.3% Triton in PBS. The experiment of immunofluorescence staining for these samples was continued as explained above (2).

Statistical analysis
The expression of PLZF in the indicated groups was calculated using one-way analysis of variance (ANOVA), continued with the Tukey's post-hoc tests (t Test) and compared with the non-parametric Mann-Whitney's test. The difference among groups was considered statistically significant if P<0.05.

Results
We first studied the localization of PLZF in the neonate and adult mouse testis (Fig.1). Immunohistochemical analysis for the cross-section of testis demonstrated that PLZF protein was expressed in the cells located on the basal membrane of adult testis seminiferous tubule, while in the neonate testis, these cells were located in the center of the tubules (Fig.1). Counting PLZF positive cells in the testis sections of the adult and neonate testis revealed significantly higher expression (P<0.05) of these cells in the adult compared to neonate (Fig.2). Furthermore, neonate and adult SSCs, ES cells and ESlike cells were cultivated in vitro, in the defined medium to investigate PLZF expression. Neonate and adult SSCs were isolated after enzyme digestion and generated cells cultivated in the presence of growth factors supporting SSC cultivation (Fig.3). Characterization of the isolated SSCs was conducted as described in our former study (2). Immunocytochemistry (ICC) analysis revealed that SSCs were positive, while pluripotent ES and ES-like cells were negative for the PLZF protein (Figs.3, 4). ES-like cell lines containing promoter-reporter Oct4-GFP transgenic mice revealed that these pluripotent cells were positive for Oct-4, but they were negative for PLZF (Fig.4). Similarly, Fluidigm real-time RT-PCR results showed significant PLZF gene expression in the neonate and 12-weeks old SSCs, compared to ES cells and ES-like cells (P<0.05, Fig.5).

Discussion
It has been demonstrated that PLZF transcription factor is a key regulator in SSCs (2). Our histological analysis specified localization of the PLZF positive cells in the center of neonatal testicular cords and basal compartment of the seminiferous tubules of adult testis, co-localized with Oct4 positive cells. PLZF/Oct4 co-localization, in a few single SSCs attached to the basal membrane, implies that these cells are SSCs, but not progenitor cells. The cultured SSCs, which are grown under GDNF stimulation, are also positive for PLZF. Although the number of PLZF positive cells in adult testis was higher than neonate, PLZF mRNA expression level in the neonate and adult SSCs was similar. Protein analyses using immunohisto/ cytochemistry revealed that PLZF was expressed in SSC, but neither in the differentiating germ cells nor in the ES-like cells directly generated from SSCs. It can be concluded that PLZF is down-regulated during both differentiation (spermatogenesis) and conversion of the unipotent SSCs into pluripotent ES-like cells. Similarly, pluripotent ES cells generated from the inner cell mass were negative for PLZF. This finding was also confirmed by Fluidigm real-time RT-PCR and ICC. These observations imply that PLZF strictly bind to and hold the molecular state of a stem cell SCCs. It is proposed that PLZF is a transcriptional repressor and activator involved in the control of SCC (29).
In undifferentiated spermatogonia, it has been shown that PLZF is co-expressed with Oct4. Mutations in the PLZF gene restrict the numbers of spermatozoa cells (9). Mutations in the PLZF display a progressive defect of SSCs and structure of the seminiferous tubule, while the function of supporting Sertoli cells is normal (20). In type A and B spermatogonia, PLZF was found to be localized in the nucleus of undifferentiated SSCs of zebrafish (30). Further studies in SSCs have indicated that the PLZF mutant shows an increase of c-Kit expression (as a marker required for differentiated SSCs), implying that PLZF maintains pool of the SSCs (19). It has been demonstrated that PLZF suppresses transcription activity of the retinoic acid receptors (31).
Although PLZF expression is positive in undifferentiated cells of stem cell compartment near the basement membrane of adult mouse testis seminiferous tubules but not in spermatocytes, it is unknown whether or not PLZF expression is necessary for initiating differentiation of the SSCs towards spermatocytes. It has been well documented that PLZF plays an important role in the self-renewal and maintenance of gonocytes and undifferentiated spermatogonia (8). PLZF has been demonstrated as a distinguished marker for the isolation of human (23,32,33), mouse (24,34) and sheep SSCs in testicular culture (35).
It is well-known that PLZF can function as both transcription activator and transcription repressor. A direct activated target of PLZF is REDD1. REDD1 mediates PLZF-dependent down-regulation of TORC1 and it is responsible for the maintenance of spermatogonial progenitor cells in culture by mediating effective signaling from GDNF, while it is normally blocked by TORC1 activity. It has been postulated that the effect of REDD1 on TORC1 could also raise the possibility that REDD1 controls cell growth, tumorigenicity and senescence (36).
PLZF activates PTEN/AKT/FOXO3 signaling pathways which can suppress prostate tumorigenesis (37). Deficiency of PLZF expression in prostate cancer is associated with tumor aggressiveness and metastasis (38). Shen et al. (39) showed that PLZF expression inhibited proliferation and metastasis via regulation of the interferon-induced protein with tetratricopeptide repeat 2 and increasing STAT1 protein level.

Conclusion
Our data demonstrated that PLZF is expressed in unipotent Oct4 + /VASA -SCCs in the basal compartment of adult testis seminiferous tubules. Our findings indicate that in comparison with unipotent SSCs, PLZF expression is not detectable in pluripotent ES-like cells which are directly derived from SCCs. Furthermore pluripotent ES cells do not express PLZF. Therefore, it could be proposed that PLZF represses and activates target genes which are specifically important for the maintenance of SSC. In the future, it would be interesting to anlyse the mechanism of PLZF down-regulation while SSCs shift to plutipotency and vice versa, during differentiation of pluripotent stem cells towards SSC in vitro. and wrote the manuscript. All authors read and approved the final manuscript.